Droplet digital isothermal nucleic acid amplification method and device based on digital microfluidics and medium

Abstract

The invention provides a droplet digital isothermal nucleic acid amplification method and device based on digital microfluidics, and a medium. The method comprises the following steps: determining the number, volume and spacing of droplets of a digital RPA reaction; designing a digital RPA liquid drop moving path; a reagent is added into the digital micro-fluidic chip, and a digital RPA reaction is completed; and performing fluorescence detection after the reaction, and calculating the copy number of the sample. The method has the characteristics of high efficiency and automation, and can rapidly and absolutely quantify the nucleic acid. The synchronous starting problem in the existing ddRPA technology is solved, the detection sensitivity and accuracy are improved, and a powerful tool is provided for future nucleic acid quantitative research.

Description

Technical Field

[0001] The present invention relates to the fields of biomedical engineering and microfluidics, and in particular to a microdroplet digital isothermal nucleic acid amplification method, equipment and medium based on digital microfluidics. Background Art

[0002] As scientific research continues to deepen, scientists are working to develop faster and more efficient nucleic acid detection methods. Recombinase polymerase amplification (RPA), as an innovative isothermal alternative to PCR, has attracted much attention since it was proposed in 2006 for its low equipment requirements (only a constant temperature environment of 37-42°C is required) and fast reaction speed (usually no more than 30 minutes). The RPA amplification method combined with microfluidics technology provides new possibilities for rapid and convenient nucleic acid detection. For example, Chinese patent CN118995974A discloses a method for rapid Helicobacter pylori detection by combining RPA with digital microfluidics, which realizes rapid multiplex detection of three target nucleic acid molecules with high sensitivity and specificity.

[0003] In the field of molecular biology research, droplet digital polymerase chain reaction (ddPCR) technology has been widely and deeply applied in many disciplines such as gene expression profiling, clinical disease diagnosis and environmental monitoring due to its excellent high sensitivity and absolute quantification capabilities. Similarly, people have also proposed a digital form of RPA method - droplet digital RPA (ddRPA), which not only guarantees high sensitivity and absolute quantification, but also has the advantages of short reaction time and low equipment requirements. For example, Chinese patent CN110575852B proposes a multi-digital RPA microfluidic chip that integrates sample pre-treatment.

[0004] However, despite the significant advantages of RPA technology, which has fast speed and low equipment requirements, it still faces severe challenges in the synchronous initiation of amplification. Unlike the hot start mechanism of PCR technology, the initiation of RPA reaction depends on the addition of Mg2+ (magnesium ions). Therefore, in the ddRPA system, the addition of Mg2+ must be performed after the droplet formation to avoid the occurrence of pre-amplification, otherwise it may lead to an overestimation of the nucleic acid concentration. At present, how to achieve effective and precise addition of Mg2+ in the ddRPA system to ensure the synchronization and accuracy of the amplification reaction is still a key issue that needs to be solved in this field. This has limited the further development and wide application of ddRPA technology to a certain extent. In order to solve the startup problem of ddRPA, researchers have proposed a variety of methods. For example:

[0005] (1) Controlling low temperature during droplet generation attempts to solve the problem, but these methods do not completely eliminate the adverse effects of pre-amplification. However, low temperature can also lead to problems such as poor droplet uniformity or even the inability to generate droplets.

[0006] Wu, X. et al. Digital CRISPR-based method for the rapid detection and absolute quantification of nucleic acids. Biomaterials 274 (2021).

[0007] Schuler, F. et al. Centrifugal step emulsification applied for absolute quantification of nucleic acids by digital droplet RPA. Lab Chip 15, 2759-2766 (2015).

[0008] (2) Using multiphase flow droplet microfluidics, where the RPA master mix and Mg2+ are injected through two inlets of a Y-shaped channel and merged into droplets at the same outlet. However, this method requires a complex microfluidic chip patterning process and has a start-up delay problem due to the sequential merging of droplets, which may lead to false negative results during the amplification process.

[0009] Cui, J. Q. et al. Droplet digital recombinase polymerase amplification (ddRPA) reaction unlocking via picoinjection. Biosensors and Bioelectronics 202 (2022).

[0010] Choi, J. W., Seo, W. H., Kang, T., Kang, T. & Chung, B. G. Droplet digital recombinase polymerase amplification for multiplexed detection of human coronavirus. Lab on a Chip 23, 2389-2398 (2023).

[0011] Liu, F. X. et al. Isothermal Background-Free Nucleic Acid Quantification by a One-Pot Cas13a Assay Using Droplet Microfluidics. Analytical Chemistry 94, 5883-5892 (2022).

[0012] (3) Use a micro-well-based dRPA microfluidic chip. During the chip patterning process, Mg2+ is pre-embedded into the micro-wells, and then the RPA master mix is pushed into the micro-wells using nitrogen gas to initiate the reaction. However, these methods also involve a complex microfluidic chip patterning process and suffer from start-up delays due to the sequential merging of droplets.

[0013] Yeh, E. C. et al. Self-powered integrated microfluidic point-of-care low-cost enabling (SIMPLE) chip. Sci Adv 3 (2017).

[0014] Shen, F. et al. Digital Isothermal Quantification of Nucleic Acids via Simultaneous Chemical Initiation of Recombinase Polymerase Amplification Reactions on SlipChip. Analytical Chemistry 83, 3533-3540 (2011). Summary of the Invention

[0015] To achieve the above objects and other advantages of the present invention, the first object of the present invention is to provide a droplet digital isothermal nucleic acid amplification method based on digital microfluidics, comprising the following steps:

[0016] Determine the number, volume, and spacing of droplets for the digital RPA reaction;

[0017] Design the movement path of digital RPA droplets;

[0018] Add reagents to the digital microfluidic chip to complete the digital RPA reaction;

[0019] After the reaction is completed, perform fluorescence detection and calculate the sample copy number.

[0020] Further, the steps of determining the number, volume, and spacing of droplets in the digital RPA reaction include:

[0021] Calculating the number and volume of droplets in the digital RPA reaction based on the estimated nucleic acid template concentration;

[0022] Assuming the interval for obtaining a preset number of droplets to obtain a droplet test array to be generated in the digital microfluidic chip;

[0023] Counting the droplet generation success rate of the droplet test array to determine an effective droplet spacing.

[0024] Further, the calculation formula for the number and volume of droplets in the digital RPA reaction is:

[0025]

[0026] Where C is the nucleic acid template concentration, with the unit of copies / μL, P is the proportion of positive droplets, P = number of positive droplets / total number of droplets; NA is the nucleic acid dilution factor, which is the multiple by which the nucleic acid template is diluted after being put into the RPA reaction system; V is the volume of a single droplet, with the unit of nL.

[0027] Further, the number of droplets is preferably 1024, and the volume of droplets is preferably 1 nL.

[0028] Further, the steps of counting the droplet generation success rate of the droplet test array to determine an effective droplet spacing include:

[0029] Calculating the ratio of the number of effective droplets to the number of generated droplets to obtain the success rate;

[0030] Judging whether the CV value of the droplet volume is less than the volume threshold to determine the number of effective droplets;

[0031] Judging whether the droplet generation success rate is greater than the success rate threshold to determine an effective droplet spacing.

[0032] Further, the preferred droplet spacing for a droplet volume of 1 nL is 600 μm, or the preferred droplet spacing for a droplet volume of 8 nL is 400 μm.

[0033] Further, the steps of designing the droplet movement path of the digital RPA include:

[0034] At the starting position of the path, two basic components are arranged: the premixed solution mother droplet and the activator mother droplet;

[0035] The premixed solution mother droplet and the activator mother droplet respectively generate their own daughter droplets;

[0036] The premixed liquid sub-droplets and the activator sub-droplets move crosswise and approach each other in a predetermined direction until, after the movement is completed, the distance between the droplets is adjusted to a preset value;

[0037] After completing the crosswise approach, the premixed liquid sub-droplets and the activator sub-droplets are paired one by one and approach each other at a closer distance to ensure that each premixed liquid sub-droplet can be in close contact with the corresponding activator sub-droplet;

[0038] By setting a specific electrode movement mode, the paired droplets are driven to carry out a mixing reaction.

[0039] Furthermore, the step of designing the digital RPA droplet movement path further includes:

[0040] By controlling the number of electrodes, the volumes of the premixed liquid mother droplet and the activator mother droplet are made equal;

[0041] The volume of each sub-droplet is precisely controlled to a preset volume corresponding to the area of the corresponding number of electrodes, and these sub-droplets are arranged in an array at a preset droplet spacing.

[0042] Furthermore, the step of adding the reagent to the digital microfluidic chip and completing the digital RPA reaction includes:

[0043] Adding the premixed solution of the nucleic acid sample to be tested and the activator containing magnesium ions to the sample loading area of the digital microfluidic platform;

[0044] Running the droplet movement path and applying a constant temperature to the digital microfluidic chip to carry out the digital RPA reaction.

[0045] Furthermore, the step of performing fluorescence detection and calculating the sample copy number after the reaction is completed includes:

[0046] Obtaining the droplets containing fluorescence labels;

[0047] Processing the original fluorescence image to create a mask;

[0048] On the basis of the mask, further demarcating the droplet area;

[0049] Calculating the fluorescence intensity of each fluorescence point within the demarcated droplet area;

[0050] Normalizing the calculated fluorescence values to distinguish positive droplets and negative droplets with a threshold;

[0051] Substituting the number of the positive droplets into the Poisson distribution formula to calculate the final nucleic acid copy number.

[0052] Furthermore, the fluorescence intensity is obtained by calculating the average gray value of the image within the demarcated droplet area.

[0053] The second object of the present invention is to provide a computer device, including a memory, a processor, and a computer program stored on the memory and executable on the processor. When the processor executes the computer program, the steps of the above method are implemented.

[0054] The third object of the present invention is to provide a computer-readable storage medium, on which a computer program is stored. When the computer program is executed by a processor, the steps of the above method are implemented.

[0055] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0056] The present invention provides a droplet digital isothermal nucleic acid amplification method, device, and medium based on digital microfluidics, which are efficient and automatic, and can quickly perform absolute quantification of nucleic acids. The present invention not only solves the synchronous start problem in the existing ddRPA technology, but also improves the detection sensitivity and accuracy, providing a powerful tool for future nucleic acid quantification research.

[0057] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly and implement it in accordance with the content of the specification, the following takes the preferred embodiments of the present invention and combines with the drawings to describe in detail as follows. The specific implementation manners of the present invention are given in detail by the following embodiments and their drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The drawings described herein are used to provide a further understanding of the present invention, form a part of this application, and the schematic embodiments and descriptions of the present invention are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0059] Figure 1 is a flowchart of a droplet digital isothermal nucleic acid amplification method based on digital microfluidics;

[0060] Figure 2 is a flowchart for determining the number, volume, and spacing of droplets in a digital RPA reaction;

[0061] Figure 3 is the lowest detection limit of a digital RPA reaction corresponding to the number of droplets N and the droplet volume V;

[0062] Figure 4 is the highest detection limit of a digital RPA reaction corresponding to the number of droplets N and the droplet volume V;

[0063] Figure 5 is a schematic diagram of electrode control of droplet volume and spacing on a digital microfluidic chip;

[0064] Figure 6 is a schematic diagram of the calculation result of the success rate of droplet generation on a digital microfluidic chip

[0065] Figure 7 To design the flow chart of the digital RPA droplet movement path;

[0066] Figure 8 Schematic diagram of the initialization stage for the design of the digital RPA droplet movement path;

[0067] Figure 9 Schematic diagram of the droplet crossing and approaching stage for the design of the digital RPA droplet movement path;

[0068] Figure 10 Schematic diagram of the droplet pairing stage for the design of the digital RPA droplet movement path;

[0069] Figure 11 Schematic diagram of the mixing reaction execution stage for the design of the digital RPA droplet movement path;

[0070] Figure 12 Flow chart for adding reagents to the digital microfluidic chip to complete the digital RPA reaction;

[0071] Figure 13 Flow chart for performing fluorescence detection after the reaction is completed and calculating the sample copy number;

[0072] Figure 14 Schematic diagram of the fluorescence change of the RPA reaction over time;

[0073] Figure 15 Schematic diagram of the OpenCv droplet recognition process;

[0074] Figure 16 Schematic diagram of a computer device;

[0075] Figure 17 Schematic diagram of a computer-readable storage medium. Specific implementation manners

[0076] Next, in combination with the accompanying drawings and specific implementation manners, the present invention will be further described. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. It should be noted that, on the premise of no conflict, any combination of the following-described embodiments or technical features may form a new embodiment.

[0077] All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0078] In this application, the accompanying drawing numbers are only used to distinguish each step in the solution and are not used to limit the execution order of each step. The specific execution order shall be subject to the description in the specification.

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. The terms used in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention.

[0080] Example 1

[0081] A droplet digital isothermal nucleic acid amplification method based on digital microfluidics, as Figure 1 shown, includes the following steps:

[0082] S1. Determine the number, volume, and spacing of droplets for the digital RPA reaction;

[0083] In some embodiments, as Figure 2 shown, the step of determining the number, volume, and spacing of droplets for the digital RPA reaction includes:

[0084] S11. Calculate the number and volume of droplets for the digital RPA reaction according to the estimated nucleic acid template concentration;

[0085] Specifically, the calculation formulas for the number and volume of droplets for the digital RPA reaction are:

[0086]

[0087] where C is the nucleic acid template concentration, with the unit of copies / μL, P is the proportion of positive droplets, P = number of positive droplets / total number of droplets; NA is the nucleic acid dilution factor, which is the multiple by which the nucleic acid template is diluted after being introduced into the RPA reaction system; V is the volume of a single droplet, with the unit of nL.

[0088] According to the above formulas, the lowest detection limit and the highest detection limit of the digital RPA reaction can be obtained, as Figure 3 and Figure 4 shown, and the detection range can be estimated therefrom.

[0089] For example, assume that a certain RPA reaction is configured with 2 μL of the original nucleic acid template, which is introduced into 48 μL of the RPA premix. At this time, the nucleic acid dilution factor is 2 / (2 + 48) = 0.04. After digitizing this RPA reaction, 1000 microdroplets with a volume of 1 nL are obtained. Substituting P = 1 / 1000 (the lowest detection limit is that only 1 droplet is positive), V = 1 nL, and KA = 0.04, the lowest detection limit of this digital RPA reaction is obtained as 25 copies / μL. Similarly, substituting P = 999 / 1000 (the highest detection limit is that 999 droplets are positive), the highest detection limit is obtained as 172694 copies / μL. Therefore, the digital RPA detection range under this configuration is 25 - 172694 copies / μL.

[0090] In some embodiments, the number of droplets is preferably 1024, and the volume of each droplet is preferably 1 nL.

[0091] The strategies for controlling droplet volume and spacing are as follows: A large number of electrode arrays are included on the digital microfluidic chip, and operations such as droplet movement and splitting are controlled by alternately applying electricity to adjacent electrodes. Different volumes and numbers of droplets can be controlled through different combinations of electrodes. For example, Figure 5 As shown, one electrode on the digital microfluidic chip in this embodiment corresponds to a square with a size of 100 μm, and the volume of the droplet controlled by one electrode is 0.25 nL. One electrode corresponds to one droplet, which is described as a 1×1 electrode; for example, a droplet occupying 4 grids is described as a 2×2 electrode, that is, 4 grids of electrodes form one droplet with a volume of 1 nL. In order to separate the droplets for digital RPA reaction, there needs to be a vacant electrode (referred to as droplet spacing) between the droplets, and the minimum unit of droplet spacing is one electrode (i.e., 100 μm).

[0092] The droplet spacing will affect the success rate of droplet generation, and the droplet spacings for different numbers and volumes of droplets are different. The droplet spacing adopted in this embodiment is determined as follows:

[0093] S12. Assume the interval for obtaining the preset number of droplets to obtain the droplet test array that needs to be generated in the digital microfluidic chip;

[0094] For example, assume that the appropriate spacing for obtaining 1000 1-nL droplets is required. It is necessary to generate a droplet test array with a volume of 1 nL and 100 - 200 droplets (10% - 20% of the number of droplets) in the digital microfluidic chip. Among them, the digital microfluidic chip and the droplet generation method belong to the prior art. For example, the digital microfluidic chips and droplet generation methods described in the patents with application numbers CN201910949813.8, CN202011552491.2, and CN202310691076.2 are not elaborated here.

[0095] S13. Statistically analyze the droplet generation success rate of the droplet test array to determine an effective droplet spacing, as Figure 6 shown.

[0096] Specifically, the step of statistically analyzing the droplet generation success rate of the droplet test array to determine an effective droplet spacing includes:

[0097] Calculate the ratio of the number of effective droplets to the number of generated droplets to obtain the success rate; that is, success rate = number of effective droplets / number of generated droplets.

[0098] Determine whether the CV value of the droplet volume is less than the volume threshold to determine the number of effective droplets. For example, if the volume threshold is set to 5%, the criterion for judging the appropriate number of effective droplets is that the CV value of the droplet volume is less than 5%.

[0099] Determine whether the droplet generation success rate is greater than the success rate threshold to determine the effective droplet spacing. For example, if the success rate threshold is set to 95%, the appropriate droplet spacing requires the droplet generation success rate to be greater than 95%.

[0100] In some embodiments, the preferred droplet spacing for a droplet volume of 1 nL is 600 μm.

[0101] In other embodiments, the preferred droplet spacing for a droplet volume of 8 nL is 400 μm.

[0102] S2. Design the digital RPA droplet movement path;

[0103] In some embodiments, as Figures 7 - 11 shown, the steps of designing the digital RPA droplet movement path include:

[0104] S21. At the starting position of the path, two basic components are arranged: the premixed solution mother droplet and the activator mother droplet;

[0105] In this embodiment, the volumes of the premixed solution mother droplet and the activator mother droplet are equal, and this equal volume distribution can be achieved by controlling the number of electrodes. For example, a 24×24 electrode array is used for precise regulation.

[0106] S22. The premixed solution mother droplet and the activator mother droplet respectively generate their own daughter droplets; among them, the digital microfluidic chip and the droplet generation method belong to the prior art. For example, the digital microfluidic chips and droplet generation methods described in the patents with application numbers CN201910949813.8, CN202011552491.2, and CN202310691076.2 are not elaborated here.

[0107] In this embodiment, the volume of each daughter droplet is precisely controlled to a preset volume, corresponding to the area of the corresponding number of electrodes, and these daughter droplets are arranged in an array at a preset droplet spacing. For example, the volume of each daughter droplet is precisely controlled to 1 nL, corresponding to the area of a 2×2 electrode, and these daughter droplets are arranged in an array at a spacing of 600 microns, as Figure 8 shown. This layout ensures sufficient space between droplets for subsequent operations while maintaining a high-density arrangement to improve processing efficiency.

[0108] S23. The premixed solution daughter droplets and the activator daughter droplets move crosswise and approach each other in a predetermined direction until, after the movement is completed, the distance between the droplets is adjusted to a preset value;

[0109] In this embodiment, the premixed liquid sub-droplets and the activator sub-droplets move crosswise and approach each other with the premixed liquid sub-droplets moving downward and the activator sub-droplets moving upward. After the movement is completed, the distance between the droplets is adjusted to 200 μm, as Figure 9 shown. This step aims to prepare for subsequent pairwise pairing and mixing reactions by precisely controlling the movement paths and spacings of the droplets.

[0110] S24. After completing the crosswise approach, the premixed liquid sub-droplets and the activator sub-droplets are paired pairwise and approach each other at a closer distance to ensure that each premixed liquid sub-droplet can be in close contact with the corresponding activator sub-droplet, as Figure 10 shown. This step is crucial to ensure the accurate and efficient progress of the mixing reaction.

[0111] S25. By setting a specific electrode movement mode, that is, adopting a reciprocating movement strategy of 2×4 electrodes, the paired droplets are driven to carry out a mixing reaction. This reciprocating movement not only promotes the full mixing between the droplets but also ensures the efficiency and uniformity of the reaction, as Figure 11 shown.

[0112] S3. Add the reagents to the digital microfluidic chip to complete the digital RPA reaction;

[0113] In some embodiments, as Figure 12 shown, the step of adding the reagents to the digital microfluidic chip to complete the digital RPA reaction includes:

[0114] S31. Add the premixed solution of the nucleic acid sample to be detected and the activator containing magnesium ions to the sample loading area of the digital microfluidic platform;

[0115] S32. Run the droplet movement path and apply a constant temperature to the digital microfluidic chip to carry out the digital RPA reaction.

[0116] In some embodiments, the premixed solution of the nucleic acid sample and the activator containing magnesium ions can be used for the RPA dual detection of influenza A / B viruses.

[0117] The total volume of the premixed solution of the nucleic acid sample is 25 μL, and the components include 14.7 μL of Buffer A, 2 μL of forward primers (1 μL of influenza A virus forward primer and 1 μL of influenza A virus reverse primer), 2 μL of reverse primers (1 μL of influenza B virus forward primer and 1 μL of influenza B virus reverse primer), 0.6 μL of exonuclease probe (0.3 μL of influenza A virus probe and 0.3 μL of influenza B virus probe), 2.5 μL of 1% F68 (used to enhance the fluidity of the reagents on the chip), 2 μL of the target nucleic acid template, and 1.2 μL of nuclease-free water.

[0118] The total volume of the activator is 25 μL, and the components include 14.7 μL of Buffer A, 2.5 μL of 1% F68, 2.5 μL of Buffer B, and 5.3 μL of nuclease-free water.

[0119] The sequences of the specific forward primer, reverse primer, and probe for influenza A / B virus are shown in the following table.

[0120]

[0121] The target nucleic acid template is used to extract RNA using a nucleic acid extraction kit according to the manufacturer's instructions. After extraction, the sample is collected in a 1.5 mL tube and stored at -80 °C until use.

[0122] In some preferred embodiments, the digital RPA temperature for influenza A / B virus detection is 39 °C.

[0123] In some preferred embodiments, obvious fluorescence can be observed 25 minutes after the start of the reaction, as Figure 14 shown.

[0124] Among the components of the premix and the activator, F68 is a non-ionic surfactant, the chemical name of which is polyoxyethylene stearate, formed by the reaction of ethylene oxide and stearic acid; the remaining components are included in common commercial RPA kits.

[0125] S4. After the reaction is completed, fluorescence detection is carried out to calculate the sample copy number. In this example, the sample copy number is calculated by the OpenCV algorithm.

[0126] In some embodiments, as Figure 13 , Figure 15 shown, the steps of fluorescence detection and calculating the sample copy number after the reaction is completed include:

[0127] S41. Obtain droplets containing fluorescent labels; for example, obtain droplets containing fluorescent labels through a fluorescence microscope or other imaging devices.

[0128] S42. Process the original fluorescence image to create a mask; this mask is used to distinguish fluorescent spots and the background in the image to more accurately identify and count fluorescent spots.

[0129] S43. Further delimit the droplet area based on the mask; this step is to determine the specific area for analysis and exclude edge effects.

[0130] S44. Calculate the fluorescence intensity of each fluorescent spot within the delimited droplet area; in this example, the fluorescence intensity is obtained by calculating the average grayscale value of the image within the delimited droplet area.

[0131] S45. Normalize the calculated fluorescence values, and distinguish positive droplets and negative droplets with a threshold value (for example, 0.2).

[0132] S46. Substitute the number of the positive droplets into the Poisson distribution formula to calculate the final nucleic acid copy number.

[0133] The present invention combines the rapid reaction ability of digital RPA and the automatic droplet manipulation ability of digital microfluidics technology to provide a rapid and automatic method for absolute quantification of nucleic acids. Compared with the 3-4-hour reaction time of traditional digital PCR, the method of the present invention can shorten the time to within 40 minutes.

[0134] Example 2

[0135] A computer device 500, as Figure 16 shown, includes a memory 510, a processor 520, and a computer program 530 stored on the memory and executable on the processor. When the processor executes the computer program, it implements the steps of a droplet digital isothermal nucleic acid amplification method based on digital microfluidics. For a detailed description of the method, reference can be made to the corresponding description in the above method embodiments, which will not be repeated here.

[0136] Example 3

[0137] A computer-readable storage medium, as Figure 17 shown, stores a computer program thereon. When the computer program is executed by a processor, it implements the steps of a droplet digital isothermal nucleic acid amplification method based on digital microfluidics. For a detailed description of the method, reference can be made to the corresponding description in the above method embodiments, which will not be repeated here.

[0138] The number of devices and the scale of processing described here are used to simplify the description of the present invention. Applications, modifications, and variations of the present invention will be apparent to those skilled in the art.

[0139] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those skilled in the art, additional modifications can be easily achieved. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the illustrated examples described here.

[0140] The device, computer device, non-volatile computer storage medium, and method provided by the embodiments of this specification are corresponding. Therefore, the device, computer device, and non-volatile computer storage medium also have beneficial technical effects similar to those of the corresponding method. Since the beneficial technical effects of the method have been described in detail above, the beneficial technical effects of the corresponding device, computer device, and non-volatile computer storage medium will not be elaborated here.

[0141] Those skilled in the art also know that in addition to implementing the controller in the form of pure computer-readable program code, the method steps can be logically programmed to enable the controller to be implemented in the form of logic gates, switches, application-specific integrated circuits, programmable logic controllers, embedded microcontrollers, etc. to achieve the same function. Therefore, such a controller can be regarded as a hardware component, and the devices included therein for implementing various functions can also be regarded as the structures within the hardware component. Or even, the devices for implementing various functions can be regarded as either software units for implementing the method or structures within the hardware component.

[0142] The systems, devices, or units illustrated in the above embodiments can be specifically implemented by computer chips or entities, or by products with certain functions. For the convenience of description, when describing the above devices, they are described as various units according to their functions. Of course, when implementing one or more embodiments of this specification, the functions of each unit can be implemented in the same or multiple software and / or hardware.

[0143] Those skilled in the art should understand that the embodiments of this specification can be provided as a method, system, or computer program product. Therefore, the embodiments of this specification can take the form of a complete hardware embodiment, a complete software embodiment, or an embodiment combining software and hardware aspects. Moreover, the embodiments of this specification can take the form of a computer program product implemented on one or more computer-usable storage media (including but not limited to disk memories, CD-ROMs, optical memories, etc.) containing computer-usable program code.

[0144] This specification is described with reference to the flowcharts and / or block diagrams of methods, devices (systems), and computer program products according to the embodiments of this specification. It should be understood that each flow and / or block in the flowchart and / or block diagram, as well as the combination of flows and / or blocks in the flowchart and / or block diagram, can be implemented by computer program instructions. These computer program instructions can be provided to the processor of a general-purpose computer, a special-purpose computer, an embedded processor, or other programmable data processing devices to generate a machine, such that the instructions executed by the processor of the computer or other programmable data processing devices generate a device for implementing the functions specified in Figure 1 one process or multiple processes and / or blocks Figure 1 one block or multiple blocks.

[0145] These computer program instructions may also be stored in a computer-readable memory capable of directing a computer or other programmable data processing device to operate in a specific manner, so that the instructions stored in the computer-readable memory produce an article of manufacture comprising an instruction device, which implements the process Figure 1 A process or multiple processes and / or boxes Figure 1 A function specified in one or more boxes.

[0146] These computer program instructions can also be loaded onto a computer or other programmable data processing device so that a series of operating steps are executed on the computer or other programmable device to produce a computer-implemented process, thereby providing instructions for implementing the process. Figure 1 A process or multiple processes and / or boxes Figure 1 The steps for the functions specified in one or more boxes.

[0147] It should also be noted that the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, commodity or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, commodity or device. In the absence of more restrictions, the elements defined by the sentence "comprises a ..." do not exclude the existence of other identical elements in the process, method, commodity or device including the elements.

[0148] The specification may be described in the general context of computer-executable instructions executed by a computer, such as program units. Generally, program units include routines, programs, objects, components, data structures, etc. that perform specific tasks or implement specific abstract data types. The specification may also be practiced in distributed computing environments where tasks are performed by remote processing devices connected via a communications network. In a distributed computing environment, program units may be located in local and remote computer storage media, including storage devices.

[0149] Each embodiment in this specification is described in a progressive manner, and the same or similar parts between the embodiments can be referred to each other, and each embodiment focuses on the differences from other embodiments. In particular, for the system embodiment, since it is basically similar to the method embodiment, the description is relatively simple, and the relevant parts can be referred to the partial description of the method embodiment.

[0150] The above are only examples of this specification and are not intended to limit one or more embodiments of this specification. For those skilled in the art, various changes and modifications can be made to one or more embodiments of this specification. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of one or more embodiments of this specification shall be included within the scope of the claims of one or more embodiments of this specification.

Claims

1. A droplet digital isothermal nucleic acid amplification method based on digital microfluidics, characterized in that: The following steps are involved: Determine the number, volume, and spacing of droplets for digital RPA reactions; Design the digital RPA droplet movement path; Adding reagents to the digital microfluidic chip to complete the digital RPA reaction; After the reaction is completed, fluorescence detection is performed to calculate the sample copy number.

2. A droplet digital isothermal nucleic acid amplification method based on digital microfluidics as claimed in claim 1, characterized in that: The step of determining the number, volume and spacing of droplets of the digital RPA reaction comprises: The number and volume of droplets in the digital RPA reaction were calculated based on the estimated nucleic acid template concentration; Assume that a preset number of droplets need to be spaced to obtain a droplet test array that needs to be generated in a digital microfluidic chip; The droplet generation success rate of the droplet test array is counted to determine the effective droplet spacing.

3. A droplet digital isothermal nucleic acid amplification method based on digital microfluidics as claimed in claim 2, characterized in that: The formula for calculating the number and volume of droplets in the digital RPA reaction is: Among them, C is the concentration of nucleic acid template, the unit is copies / μL, P is the proportion of positive droplets, P = number of positive droplets / total number of droplets; NA is the nucleic acid dilution factor, the multiple of dilution of the nucleic acid template after being put into the RPA reaction system; V is the volume of a single droplet, the unit is nL.

4. A droplet digital isothermal nucleic acid amplification method based on digital microfluidics as claimed in claim 2, characterized in that: The number of droplets is preferably 1024, and the droplet volume is preferably 1 nL.

5. A droplet digital isothermal nucleic acid amplification method based on digital microfluidics as claimed in claim 2, characterized in that: The step of counting the droplet generation success rate of the droplet test array to determine the effective droplet spacing includes: The ratio of the number of effective droplets to the number of generated droplets is calculated to obtain the success rate; Determine whether the droplet volume CV value is less than the volume threshold to determine the effective number of droplets; It is determined whether the droplet generation success rate is greater than a success rate threshold to determine the effective droplet spacing.

6. A droplet digital isothermal nucleic acid amplification method based on digital microfluidics as claimed in claim 5, characterized in that: The preferred droplet spacing for a droplet volume of 1 nL is 600 μm, or the preferred droplet spacing for a droplet volume of 8 nL is 400 μm.

7. A droplet digital isothermal nucleic acid amplification method based on digital microfluidics as claimed in claim 1, characterized in that: The step of designing the digital RPA droplet movement path includes: At the beginning of the path, the arrangement has two basic components: a premix mother droplet and an activator mother droplet; The premix mother droplet and the activator mother droplet respectively generate their own daughter droplets; The premixed liquid sub-droplet and the activator sub-droplet move in a cross-approaching manner in a predetermined direction until the distance between the droplets is adjusted to a preset value after the movement is completed; After the cross approach is completed, the premixed liquid sub-droplets and the activator sub-droplets are paired in pairs and approach each other at a closer distance, ensuring that each premixed liquid sub-droplet can be in close contact with the corresponding activator sub-droplet; The paired droplets are driven to undergo mixing reactions by setting a specific electrode movement pattern.

8. A droplet digital isothermal nucleic acid amplification method based on digital microfluidics as claimed in claim 7, characterized in that: The step of designing a digital RPA droplet movement path also includes: The volume of the premix mother liquid droplet and the volume of the activator mother liquid droplet are equal by controlling the number of electrodes; The volume of each sub-droplet is precisely controlled to a preset volume, corresponding to the area of ​​a corresponding number of electrodes, and the sub-droplets are arranged in an array at a preset droplet spacing.

9. A droplet digital isothermal nucleic acid amplification method based on digital microfluidics as claimed in claim 1, characterized in that: The step of adding the reagent to the digital microfluidic chip to complete the digital RPA reaction includes: Adding a premixed solution of a nucleic acid sample to be tested and an activator containing magnesium ions to a sample loading area of ​​a digital microfluidics platform; Run the droplet movement path and apply a constant temperature to the digital microfluidic chip to perform digital RPA reaction.

10. A droplet digital isothermal nucleic acid amplification method based on digital microfluidics according to claim 1, characterized in that: After the reaction is completed, fluorescence detection is performed, and the step of calculating the sample copy number includes: Obtaining droplets containing fluorescent markers; The original fluorescence image was processed to create a mask; Based on the mask, further delineating the droplet area; Calculate the fluorescence intensity of each fluorescent point within the defined droplet area; The calculated fluorescence values ​​were normalized and the positive droplets were distinguished from the negative droplets by the threshold value; The number of positive droplets is substituted into the Poisson distribution formula to calculate the final nucleic acid copy number.

11. A droplet digital isothermal nucleic acid amplification method based on digital microfluidics according to claim 10, characterized in that: The fluorescence intensity is obtained by calculating the average gray value of the image within the defined droplet area.

12. A computer device comprising a memory, a processor and a computer program stored in the memory and executable on the processor, characterized in that: When the processor executes the computer program, the steps of the method according to any one of claims 1 to 11 are implemented.

13. A computer-readable storage medium having a computer program stored thereon, characterized in that: When the computer program is executed by a processor, the steps of the method according to any one of claims 1 to 11 are implemented.

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